Lymphoid tissue, like many others, depend upon a balance between cellular input and output in order to maintain homeostasis and prevent proliferation-related disorders. Frequently, this balance represents equilibrium between cell division and programmed cell death (PCD). A linkage between PCD and the cell division cycle has become obvious in recent years. The hypothesis put forth in this application, i.e., that Bcl-2 may regulate PCD through regulation of the cell division cycle, suggests a direct linkage for the regulation of these two processes, and has implications for both. It has recently been shown that Bcl-2 inhibits the G1/S phase transition of cell cycle, in vivo and in vitro, in a variety of cell types. This finding is appealing because the G1/S phase transition is a known control point from which cell division and cell death pathways diverge. Since G1/S phase effects are now confirmed, the experiments proposed in the current application are directed at understanding the mechanism by which Bcl-2 might regulate this transition. The goal of the proposed studies is to provide an understanding of the biological consequences of Bcl-2 regulation of cell cycle, including the regulation of PCD and the propagation of malignant transformations. The goals will be approached in a variety of ways. One will be to identify the Bcl-2 protein domains which may be critical in cell cycle regulation, versus those which are known to be important in the direct antagonism of cell death mediators (i.e., Bax). This will be performed by site-directed mutagenesis or amino acid substitution in existing Bcl-2 expression vectors, followed by analysis of the cell cycle/cell death effects of these mutations in transfected cell lines. The protein domain search will be limited by first evaluating the cell cycle regulatory effects of other members of the Bcl-2- family, then concentrating on domains which are similar in family members that do have cycle regulatory effects, or on domains which are unique to Bcl-2 in those that do not. In addition to identifying the protein domains which are active in cell cycle regulation, the biochemical mechanism of the cell cycle effect will be sought. Since differential phosphorylation of the retinoblastoma protein (pRb) in the presence or absence of Bcl-2 has been demonstrated, and since pRb phosphorylation is a critical control point in the G1/S phase transition, efforts will center around this finding. Investigations will proceed both upstream and downstream of pRb, by attempting to determine how pRb phosphorylation is regulated by Bcl-2, and by determining how such an effect could alter the cell division/cell death decision.